Hepatitis E Virus (HEV) is a leading cause of severe acute hepatitis worldwide and numerous outbreaks in the developing countries. The virus is considered to be an 'emerging infectious agent' because of its propensity to infect humans, livestocks and wild animals, and its ability to cross 'species-barriers'. The replication system described so far are not efficient and does not permit visualization of replication products such as negative strand RNA, subgenomic RNAs and replicative forms/intermediates (RFs/Rls) by gel electrophoresis. This proposal is designed to develop numerous cell lines with ability to support constitutive replication of selectable self-replicating RNAs (replicons) that are derived from a full-length HEV clone. These HEV replicons encode selectable Neo gene that will permit selection and expansion of only those cells which support efficient replication of HEV. An EMCV IRES in the replicon drives efficient translation of viral proteins under normal as well as adverse situations when cap-dependent translation of cellular mRNAs is shut-off. The cytoplasmic lysates derived from cell lines expressing replicons will be used to develop a robust cell-free HEV replication system. An enhanced green florescence protein (EGFP) system has also been placed in a HEV replicon to directly visualize replication process in the cells. We have described several alternative approaches to achieve the goals of developing efficient HEV replication system. The experiments are designed to characterize replicon-based expression of viral proteins and the replication products. This method will help in dissecting the mechanism of HEV infectious processes and will also help in understanding HEV biology. The cell-free system described here further offers opportunity to screen compounds with anti-HEV activities. [unreadable] [unreadable] [unreadable]